Journal of Experimental Botany, Vol. 48, No. 307, pp. 229-237, February 1997Journal ofExperimentalBotany

Identification of G proteins mediating fungalelicitor-induced dephosphorylation ofhost plasma membrane H-ATPase

Ti Xing, Verna J. Higgins and Eduardo Blumwald1

Department of Botany, University of Toronto, 25 Willcocks St., Toronto, Ontario M5S 3B2, Canada

Received 19 March 1996; Accepted 12 August 1996

Abstract

Tomato cells (with the Cf-5 resistance gene) weretreated with elicitor preparations containing the avr5gene product from two Cf-5 incompatible races of thefungal pathogen Cladosporium fulvum (race 2.3 andrace 4), or with elicitor preparations containing no avr5gene product from two Cf-5 compatible races (race 5and race 2.4.5.9.11). Elicitor preparations from race2.3 or race 4 caused dephosphorylation of host plasmamembrane H + -ATPase in isolated plasma membranes,while the preparations from race 5 or race 2.4.5.9.11did not. GTP(y)S, AIF4", and cholera toxin (CTX) eachinduced similar dephosphorylation in the absence ofactive elicitors. The elicitor-induced dephosphoryl-ation of the H+-ATPase was blocked by preincubationof membranes with an antibody raised against a stimul-atory G protein ce-subunit (anti-Gsu). This antibodycross-reacted with a 42 kDa polypeptide from tomatoplasma membranes. A 42 kDa polypeptide was alsoADP-ribosylated by CTX. When plasma membraneswere treated with elicitor preparations from race 4and separated on non-dissociating PAGE, two proteinswere detected on Western blots with the antibodyraised against the a-subunit, suggesting the dissoci-ation of the trimeric complex. No dissociation of thecomplex was detected with antibodies raised againsteither the a- or /7-subunits when the plasma mem-branes were treated with elicitor preparations fromrace 5. The results provide evidence for the activationof a stimulatory subtype of trimeric G proteins in thestimulation of elicitor-induced host defences to fungalpathogens.

Key words: G protein, dephosphorylation, H+-ATPase,fungal elicitor, tomato.

Introduction

GTP-binding proteins (G proteins) have the ability tobind guanosine triphosphate (GTP) and hydrolyse it toguanosine diphosphate (GDP). G proteins act as molecu-lar signal transducers where active or inactive statesdepend on the binding of GTP or GDP, respectively.Receptor activation by ligands enhances GTP binding tothe G protein, and the activated G protein then selectivelymodifies the activity of the effector (Gilman, 1987; Kiziroet al., 1991). The G proteins include two major subfamil-ies, the heterotrimeric G proteins and the small G pro-teins. Whereas the heterotrimeric G proteins contain a-,j8- and y- subunits, the small G proteins appear to besimilar to free a-subunits, operating without the py-subunits. The heterotrimeric family contains differenttypes of G proteins, e.g. Gs (stimulatory G proteins), Gj(inhibitory G proteins), G, (transducins). Based on thesimilarity in amino acids and the presumed function,small G proteins can be grouped into five subtypes: Ras/Ras-like, Ran/TC4, Rho/Rac, Rab/Ypt, and Arf/Sar(Verma et al., 1994). Generally, it is the a-subunit of theG protein which has the receptor and effector bindingregions and which possesses a guanosine nucleotide bind-ing site and GTPase activity (Gilman, 1987; Kaziro et al.,1991). Both classes of G proteins use the GTP/GDP cycleas a molecular switch for signal transduction. Interactionof the G protein with the activated receptor promotes theexchange of GDP, bound to the a-subunit, for GTP andthe subsequent dissociation of the a-GTP complex fromthe jSy-heterodimer. A single receptor can activate multipleG protein molecules, thus amplifying the ligand bindingevent.

The existence and the possible function for G proteinsin plants has been increasingly documented. G proteins

1 To whom correspondence should be addressed. Fax: +1 416 978 5878. E-mail; blumwald@botany.utoronto.ca

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have been shown to be involved in the control of K+

channel opening in guard cells and mesophyll cells(Fairley-Grenot and Assmann, 1991; Wu and Assmann,1994), the transmission of red and blue light-inducedsignals (Warpeha et al, 1991; Neuhaus et al, 1993) andthe pathogen-induced resistance reactions (Legendreet al, 1992). However, identification of the subtypes ofG proteins that mediate particular G protein-mediatedbiochemical and physiological processes has lagged,especially in the case of trimeric G protein-mediatedprocesses (Terryn et al, 1993). Tomato Cf-9 gene, confer-ring resistance to infection by races of the fungus C.fulvum that carry the avirulence gene Avr9, has beenisolated, and the predicted protein showed homology tothe receptor domain of several receptor-like kinases(RLK) in Arabidopsis (Jones et al, 1994). The identifica-tion of downstream elements in pathogen signal transduc-tion, including G proteins, will significantly enhanceunderstanding of the resistance mechanisms.

Race-specific resistance of tomato to the fungal patho-gen C. fulvum involves a gene-for-gene relationshipbetween host and fungus. Specific recognition in thisinteraction is mediated via specific fungal elicitors (DeWit, 1992). Treatment of tomato cell suspension culturewith intercellular fluid (IF) obtained from infected plantsand containing the appropriate specific elicitor inducedthe dephosphorylation of plasma membrane H + -ATPasewith the concomitant increase in enzyme activity (Vera-Estrella et al, 1994a; Xing et al, 1996) and initial studiessuggested the involvement of G proteins (Vera-Estrellaet al, 1994a). In this study, the involvement of G proteinswas further investigated and a subtype of G protein thatfunctions in the dephosphorylation of host plasma mem-brane H+-ATPase was identified.

Materials and methods

Plant material

Tomato cell suspensions derived from a line of tomato(Lycopersicon esculentum L. cv. Moneymaker) with the resistancegene Cf-5, were grown in 500 ml Erlenmeyer flasks containing100 ml of MS medium (Murashige and Skoog, 1962), in thedark at 25 °C on a rotatory shaker at 120 rpm and subculturedweekly (Vera-Estrella et al, 1992). Cells suspension used forall experiments were 3-4-d-old.

Production of elicitors

Intercellular fluids (IF) from tomato leaf tissue infected withCladosporium fulvum were prepared according to the method ofDe Wit and Spikman (1982), using cv. Bonny Best (no knownCf genes) inoculated with the Cf-5 incompatible races 2.3 and4, or compatible races 5 and 2.4.5.9.11. The IFs obtained bycentrifugation of water-infiltrated infected leaves were precipit-ated with acetone (90%) and stored at — 20 °C overnight. Thepreparations were centrifuged at 1650 g for 20 min, thesupernatant discarded and the pellet resuspended in distilledwater and boiled for 30 min followed by centrifugation at 1650

g for 20 min. (Although boiling causes some loss of activity ofthe avrS elicitor based on an assay for the oxidative burst, itprecipitates considerable contaminating protein.) The pellet wasdiscarded and the supernatant was freeze-dried. Distilled waterwas added to give the original volume and the preparationswere stored at — 20 °C. These preparations contained specificelicitors for each of the Cf genes on which the specific race ofC. fulvum is avirulent (i.e. causes a hypersensitive response)(De Wit and Spikman, 1982) and this specificity was alsoshown for plasma membrane-associated activities, such as H + -ATPase, redox, etc. (Vera-Estrella et al, 1994a, b). Generally,a ratio of IF to water of 1/32 was used and this containedbetween 0.10-0.45 /xg of protein /xl"1 as measured according toBradford (1976). Once specificity of IFs was established for thedephosphorylation reaction, for several experiments no IF orrace 4 IF at 1:160 dilution served as controls.

Plasma membrane isolation

Microsomal fractions from Cf-5 cell suspensions were isolatedas described before (Vera-Estrella et al, 1994a). Microsomeswere resuspended in 0.2 ml of phase suspension mediumcontaining 250 mM sucrose, 5 mM potassium phosphate buffer(pH7.0) and 1 mM DTT. The microsomes were phase-partitioned according to Thomson et al. (1993) in an 8 g systemwith a final composition of 6.5% (w/w) Dextran T500, 6.5%(w/w) PEG (mol. wt. 3350), 250 mM sucrose, 3 mM KC1,5 mM potassium phosphate (pH 7.8), and 1 mM DTT. Theresulting upper phase was partitioned through two additionalsteps, and the upper phase from the third partition step wasdiluted 10-fold in phase-diluting medium containing 250 mMsucrose, 25 mM K2SO4, 20 mM TR1S-MES (pH 7.0), and1 mM DTT. The membranes were collected by centrifugationat 100 000 g for 40 min at 4°C and resuspended in suspensionbuffer containing 250 mM sucrose, 10% (v/v) glycerol, 1 mMDTT, and 2 mM TRIS-MES (pH7.0). The phase-purifiedplasma membranes were either used immediately, or frozen inliquid nitrogen and stored at — 70 °C until use. The plasmamembranes were leaky enough to allow the reagents to reachthe vesicular lumen.

In vitro phosphorylation assays

Plasma membrane protein was incubated at 25 °C for 15 minin phosphorylation buffer containing 10% glycerol, 6 mM TRIS-MES (pH6.5), 2mM DTT, 250 mM mannitol, 1 pM GTP,5mM MgSO4, 5mM KC1, and I0>Ci of y-32P-ATP. IFpreparations were added to give a final dilution of 1:32 andafter incubation for 15 min at 25 °C, the reaction was stoppedby the boiling step in the immunoprecipitation (see below).Proteins measured by the Bradford method (Bradford. 1976)were then immunoprecipitated.

Immunoprecipitation, SDS-PAGE and autoradiography

Following the in vitro phosphorylation assays, plasma membraneproteins were immunoprecipitated using antibodies raisedagainst the N-terminal of the 100 kDa plasma membrane H + -ATPase isolated from Arabidopsis thaliana (a gift from RamonSerrano, Spain). For immunoprecipitation of the plasmamembrane H + -ATPase, 15-25 /xg of isolated plasma membranewere boiled for 5 min in 2 x immunoprecipitation buffer (20 mMNa phosphate, pH 7.2, 300 mM NaCl, 2 mM EDTA, and 3%Triton X-100), followed by centrifugation for 10 min at 15 000g. An equal volume of 2 x immunoprecipitation buffer wasadded to the supernatant together with 5 yA of anti-H+-ATPase.After 10 min of gentle mixing, they were incubated overnightat 4 °C. An equal volume of 10% pre-washed protein-A-agarose

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was added. (Protein-A-agarose was washed with 50 vols of1 x immunoprecipitation buffer by mixing and centrifugation.and resuspended in immunoprecipitation buffer giving a finalconcentration of 10%.) The samples were incubated at roomtemperature for 2 h before centrifugation for 4 min at 15 000 gin a microcentrifuge. The supernatant was discarded and thepellet was washed three times with 1 ml immunoprecipitationbuffer by mixing and centrifugation. The pellet was finallyresuspended in SDS-PAGE sample buffer (Laemmli, 1970),centrifuged at 15 000 g, and the supernatant used for SDS-PAGE. Proteins were separated using a discontinuous SDS-PAGE (12.5% separation gel) system and the pattern ofphosphorylated polypeptides was visualized by autoradiographyusing Kodak X-Omat AR autoradiography film.

Western immunoblotting

Western immunoblotting was performed using the anti-H+-ATPase or antibodies raised against the a-subunit of astimulatory G protein (Gsa) or the a-subunit of inhibitory Gproteins (Gm-1, Gia-2, Gial-3) or the a-subunit of the Go-type ofG proteins or against the /3-subunit.

In vitro ADP-ribosylation with CTX

In vitro ADP-ribosylation was conducted according to Gill andWoolkalis (1991) with some modifications. Plasma membraneswere diluted in 1% Lubrol PX (Pierce), 10 mM TRIS-HC1(pH 7.5) and 0.1 mM EDTA to a protein concentration of1-3 /̂ g (A'1. Twenty fA aliquots of membranes were assayed ina final volume of 50 fA containing 5 u.M 32P-NAD, 250 mMpotassium phosphate buffer (pH 7.0), 20 mM thymidine, 3 mMATP, 3mM GTP, 0.1 mM GPP(NH)P, and 20 mM argininehydrochloride. The toxin was added at a final concentration of50 ^g ml" ' . (CTX was activated by treatment with 50 mM DTTfor 60 min at room temperature prior to assay.) The assay wasinitiated by transfer to a 37 °C water bath for 60 min and thenterminated by transferring to ice. Proteins were denatured withLaemmli (1970) buffer without heating and separated on 12.5%SDS-PAGE. ADP-ribosylated proteins were detected byautoradiography.

Elicitor-induced dissociation of G protein subunits

Plasma membrane protein (usually 100-150 ug) was incubatedat 25 CC for 15 min in phosphorylation buffer except that they-32P-ATP was omitted. After a further 15 min incubation with,or without, the addition of IF preparations at 25 °C, aliquotswere supplemented with 50 mM KC1 and 0.1 mM EGTA andrepeatedly (three times) frozen in liquid N2 and thawed in awater bath at 20 °C. This process produced a mixture of inside-out and outside-out vesicles. The formation of inside-outvesicles was traced by monitoring non-latent ATPase activity(Larsson el cil., 1994). The membrane vesicles were than washedwith 5 vols of TED buffer (20 mM TRIS-HC1, pH 8.0, 1 mMEDTA, and 1 mM DTT) plus 300 mM NaCl prior to extraction(Northup et al., 1980; Birnbaumer et al., 1994). After collectionby centrifugation, the membranes were suspended to a finalvolume of 50 ^1 of TED containing 50 mM NaCl and 1.0%sodium cholate and extracted for 60 min by gentle shaking at4°C. Membranes were then removed by centrifugation for 75min at 110 000 g. Aliquots of supernatant were used for 10%non-denaturating discontinuous PAGE (Hames, 1990).Following electrotransfer, the nitrocellulose was probed withthe polyclonal antibody against Gstl of human origin.

Gs activation by fungal elicitors 231

Chemicals

y-32P-ATP and y-32P-NAD were purchased from AmershamInternational. Anitbodies raised against the a-subunit of astimulatory G protein (Gsa) were from Santa CruzBiotechnology (Santa Cruz, USA). Other antibodies (anti-G1CI-1, anti-Gio-2, anti-Gia-3, anti-Gocx, and anti-Gp), choleratoxin (CTX), pertussis toxin (PTX), and toxin C3 werepurchased from CalBiochem (San Diego, USA). Other commonchemicals were purchased either from ICN Biomedicals(Mississauga, Ontario, Canada), Sigma (St Louis, USA) orBDH Inc. (Toronto, Ontario, Canada).

Results

Specificity of the dephosphorylation of the host plasmamembrane H+-ATPase

Previous studies with Cf-5 cell suspension cultures sug-gested that the recognition of an elicitor from C. fulvumby putative receptors at the host plasma membrane,resulted in the induction of signal transduction pathwaysthat initiated the defence reponses against the pathogen(Vera-Estrella et al., 1994a, b; Xing et al., 1996). One ofthese signals invoked a rapid dephosphorylation of theplasma membrane H+-ATPase (Vera-Estrella et al.,1994a; Xing et al., 1996). It has been shown that elicitorsfrom race 4 (but not from race 5) of C. fulvum induceddephosphorylation in vitro within 30 min (Xing et al.,1996). Here, the race specificity of elicitor-induceddephosphorylation of the H +-ATPase was further con-firmed by treatment of plasma membranes (isolated fromtomato cells containing the Cf-5 gene) for 15 min withIFs obtained from two Cf-5 incompatible races of C.fulvum (races 2.3 and 4) and two Cf-5 compatible races(race 5 and 2.4.5.9.11). The treatment of plasma mem-branes with race 2.3 or race 4 IFs resulted in the dephos-phorylation of the H+-ATPase (Fig. 1A, lanes 2 and 3),identified as a 100 kDa polypeptide by immunoblotting(Fig. IB). However, no dephosphorylation was observedwhen the membranes were treated with race 5 IF or race2.4.5.9.11 IF (Fig. 1 A, lanes 4 and 5).

Effect of GTP(y)S, fluoride and bacterial toxins onelicitor-induced dephosphorylation of the host plasmamembrane H+-ATPase

The involvement of G proteins mediating the elicitor-induced dephosphorylation of the host plasma membraneH+-ATPase was indicated by the effects of GTP(y)S[guanosine 5'-O-(3 thio) triphosphate] and A1F4~ ions(Fig. 2). The non-hydrolysable guanosine nucleotide ana-logue GTP(y)S locks G proteins in a GTP-bound activeform (Kaziro et al., 1991). GTP(y)S promoted thedephosphorylation of the plasma membrane H+-ATPasein the absence of an active elicitor or in the presence ofa 1:160 elicitor dilution, treatments in which dephos-phorylation does not normally occur (Fig. 2A, lanes 4

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Fig. 1. In vitro y-32P-ATP-labelled plasma membrane proteins aftertreatment with elicitor preparations from races of C. fulvum differingin specificity on Cf-5 plants. (A) Specificity of dephosphorylation: cellswere grown for 4 d and plasma membranes isolated. Plasma membraneswere incubated for 15 min in the phosphorylation reaction mixture andIFs from different races were then added. Labelling was stopped byboiling during immunoprecipitation. The samples were immunoprecipit-ated followed by separation on SDS-PAGE and the bands viewed byautoradiography. Lane 1, no IF; lanes 2 and 3, 1:32 diluted IF fromCf-5 incompatible races 2.3 and 4, respectively; lanes 4 and 5, 1:32diluted IF from Cf-5 compatible races 5 and 2.4.5.9.11, respectively.(B) Recognition of a polypeptide by the antibody directed against H + -ATPase. Untreated plasma membranes were probed with an antibodyraised against the 100 kDa plasma membrane H+-ATPase fromArabidopsis thaliana in immunoblotting. The arrow represents a 100 kDapolypeptide, which cross-reacted with the antibody raised against theH + -ATPase. Numbers on the left represent molecular masses of proteinstandards in kDa. The gel represents five independent experiments.

and 5). A1F4 ions (obtained by using NaF in thepresence of A1C13) are a useful tool for the identificationof G protein-induced processes because they can mimicGTP analogues (Higashijima et al., 1987). The incubationof tomato plasma membranes with A1F4" resulted in thedephosphorylation of the plasma membrane H+-ATPasein the absence of active elicitors or in the presence of adilution of the elicitor (1:160) that does not inducedephosphorylation (Fig. 2B, lanes 4 and 5).

Several potent bacterial toxins exert their effects byselectively inducing the ADP-ribosylation of certain G

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Fig. 2. Effects of race 4 IF, GTP(y)S, or AlF4~ on the dephosphoryl-ation of the plasma membrane H + -ATPase. (A) GTP(y)S:Autoradiogram of m vitro y-32P-ATP-labelled plasma membraneproteins showing the effect of race 4 IF or GTP(y)S on thedephosphorylation of host plasma membrane H+-ATPase. After 15min in the phosphorylation reaction mixture, different treatments wereapplied. In vitro labelling was carried out in the absence of IF (lane 1),in the presence of 1:32 diluted race 4 IF (lane 2), 1:160 diluted race 4IF (lane 3), 1:160 diluted race 4 IF plus 100 ^M GTP(y)S (lane 4) or100ftM GTP(y)S only (lane 5). Labelling was stopped with the boilingprocess in immunoprecipitation. The samples were immunoprecipitatedfollowed by separation on SDS-PAGE and the bands viewed byautoradiography. (B) A1F4": Autoradiogram of in vitro y-32P-ATP-labelled plasma membrane proteins showing the effect of race 4 IF orA1F4" (obtained by adding 20 mM NaF and 50 mM A1C13 to thephosphorylation buffer) on the dephosphorylation of host plasmamembrane H + -ATPase. After 15 min in the phosphorylation reactionmixture, different treatments were applied. In vitro labelling was carriedout in the absence of IF (lanel), in the presence of 1:32 diluted race 4IF (lane 2), 1:160 diluted race 4 IF (lane 3), 1:160 diluted race 4 IFplus 20mM NaF and 50mM A1C13 (lane 4) or 20mM NaF and50mM A1C13 only (lane 5). Labelling was stopped with the boilingprocess in immunoprecipitation. The samples were immunoprecipitatedfollowed by separation on SDS-PAGE and the bands viewed byautoradiography. The arrow represents a 100 kDa polypeptide, whichcross-reacted with the antibody raised against the H+-ATPase. Numberson the left of each panel represent molecular masses of proteinstandards in kDa. Gels represent three independent experiments.

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Gs activation by fungal elicitors 233

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Fig. 3. Effect of bacterial toxins on the dephosphorylation of hostplasma membrane H + -ATPase. Autoradiogram of in vitro y-32P-ATP-labelled plasma membrane proteins. In vitro labelling was carried outin the absence of IF (lane 1), in the presence of 1:32 diluted race 4 IF(lane 2), 1:160 diluted race 4 IF (lane 3), 1:160 diluted race 4 IF plus500ngmr' CTX (lane 4) or in 500 ng ml"1 CTX only (lane 5), 1:160diluted race 4 IF plus 500ngmr' PTX (lane 6) or in 500ngml"'PTX only (lane 7), 1:160 diluted race 4 IF plus 500 ng ml"1 toxin C3(lane 8) or in 500ngml~' toxin C3 only (lane 9). 5 ^M NAD wasadded as the substrate for ADP-ribosylation of G proteins. The sampleswere immunoprecipitated followed by separation on SDS-PAGE andthe bands viewed by autoradiography. The arrow represents a 100 kDapolypeptide, which cross-reacted with the antibody raised against theH+-ATPase. Numbers on the left of each panel represent molecularmasses of protein standards in kDa. The gel represents three independentexperiments.

proteins, inducing their activation (Dolphin, 1987). Theeffects of three of these toxins on elicitor-induced dephos-phorylation of host plasma membrane H+-ATPase wereinvestigated (Fig. 3). In the presence of CTX (from Vibriocholerae), the dephosphorylation of the enzyme wasinduced in the absence of active elicitors (no IF or 1:160dilution) (Fig. 3, lanes 4 and 5). Dephosphorylation inthese treatments did not occur if CTX was replaced bypertussis toxin (PTX, from Bordetella pertussis) or toxinC3 (from Clostridium botulinum) (Fig. 3, lanes 6-9).

In vitro ADP-ribosylation with CTX

Membrane preparations were incubated with y-32P-NAD,followed by SDS-PAGE and autoradiography. A 42 kDapolypeptide was labelled by y-32P-NAD in the presenceof both plasma membrane and CTX (Fig. 4, lane 3).There was no intrinsic ribosylation as indicated by thetreatment with plasma membrane only (Fig. 4, lane 1).Excess of unlabelled NAD (lOO^M) completely elimin-ated the 32P-labelling (Fig. 4, lane 2) thus indicating thatthe 32P-labelling of the 42 kDa polypeptide was due toenzymatic transfer of ADP-ribose from y-32P-NAD. TheADP-ribosylation was inhibited by pre-incubation of themembranes with an antibody which was highly selectiveagainst GM IgG (Fig. 4, lane 4). No ribosylation ofplasma membrane proteins were observed when plasmamembranes were treated with PTX or toxin C3 (datanot shown).

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Fig. 4. ADP-ribosylation of plasma membrane proteins. Plasma mem-branes were diluted in l% Lubrol PX, lOmM TRIS-HCl, 0.1 mMEDTA, pH 7.5, to a protein concentration of 1-3/xg jd"1. ADP-rybosylation conditions were as described in Materials and methods.Lane 1, plasma membrane only; lane 2, 100 juM unlabelled NAD, CTXand plasma membrane; lane 3, CTX and plasma membrane; and lane4, CTX and plasma membrane which was pre-incubated for 15 minwith anti-G^ IgG (1:10 dilution). The arrow indicates the ribosylatedband. Numbers on the left represent molecular masses of proteinstandards m kDa. The gel represents three independent experiments.

Blockage of elicitor-induced dephosphorylation of the hostplasma membrane H+-ATPase by antibodies raised againstdifferent G protein subunits

To identify the subtype of G proteins, several highlyselective commercial antibodies to the a-subunits ofdifferent heterotrimeric G proteins were used to block theelicitor-induced dephosphorylation of the H+-ATPase.These included anti-Gia-l, anti-Gia-2, anti-Gloe-3, anti-Goot,and anti-Gsa. Incubation with anti-Gsot inhibited thedephosphorylation of plasma membrane H+-ATPase(Fig. 5A, lane 5), whereas other antibodies did not(Fig. 5A, lanes 3 and 4). The Gsot antiserum cross-reactedwith a 42 kDa polypeptide in immunoblotting (Fig. 5B).The similarity in the apparent molecular mass of thepolypeptide that was ADP-ribosylated by CTX (Fig. 4)and the polypeptide that cross-reacted with the anti-GS0(

(Fig. 5B) would suggest that this polypeptide belongs tothe stimulatory subtype of G proteins.

Elicitor-induced dissociation of G protein subunits

For the isolation of G proteins, plasma membrane pre-parations were incubated for 10 min with IF preparationsin phosphorylation buffer lacking y-32P-ATP. The mem-brane vesicles were then frozen and thawed three timesto produce a mixture of inside-out and outside-out vesiclesin order to expose G proteins (including the dissociatedand the non-dissociated forms) to the buffer. These ves-icles were then gently solubilized to release G proteinsfrom the membrane without altering their subunit attach-ment (Northup et ai, 1980; Birnbaumer et al., 1994).The solubilized fraction was applied to native PAGE

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Fig. 6. Elicitor-induced dissociation of G protein subunits detected withanti-Gso and anti-Gp. (A) Anti-GSCI. Plasma membrane vesicles weretreated for 15 min with no IF (lane 1), with race 4 IF (lane 2) or withrace 5 IF (lane 3). The membrane vesicles were frozen and thawedfollowed by buffer washing as described in Materials and methods. Thesolubilized fractions were separated on 10% non-dissociating discontinu-ous PAGE. Following electrotransfer, the proteins were detected witha polyclonal antibody raised against Gsa. Arrows indicate the cross-reacted bands. (B) Anti-Gp Plasma membrane vesicles were treated asdescribed in (A) with the same lane settings. Following electrotransfer.the proteins were detected with a polyclonal antibody raised againstGp. Arrows indicate the cross-reacted bands. Gels represent fourindependent experiments.

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Fig. 5. Effect of different G protein antibodies on the dephosphorylationof host plasma membrane H + -ATPase. (A) Blockage of dephosphoryl-ation by antibodies raised against different G proteins. Autoradiogramof in vitro y-32P-ATP-labelled plasma membrane proteins. Antibodieswere incubated with plasma membrane (suspended in 1% Lubrol PX)at 1:10 dilution for 15 min in phosphorylation buffer prior to m vitrolabelling. In vitro labelling was carried out in the absence of IFfollowing pre-incubation with H2O (lane 1), in the presence of 1:32diluted race 4 IF following pre-incubation with H2O (lane 2), anti-Gio-1 and G,a-2 (lane 3), anti-Gm-3 and Goa (lane 4) or anti-G5!r (lane5). The samples were immunoprecipitated followed by separation onSDS-PAGE and the bands viewed by autoradiography. The arrowrepresents a 100 kDa polypeptide, which cross-reacted with the antibodyraised against the H + -ATPase. (B) Recognition of a polypeptide by theantibody directed against the C-terminal of G,a subunit. Membraneproteins were separated by SDS-PAGE and immunoblots were per-formed as descibed in Matenals and methods with the antibody raisedagainst the C-terminal of G50, subunit. The arrow indicates the cross-reacted band. Numbers on the left of each panel represent molecularmasses of protein standards in kDa. Gels represent three independentexperiments.

followed by immunoblotting. Two bands, presumably thea/3y-trimeric complex and the a-subunit were detected(Fig. 6A, lane 2) with an antibody against Gsa whenmembranes were treated with race 4 IF. Only the trimericcomplex was detected in treatments with no IF or withrace 5 IF (Fig. 6A, lane 1 and 3). When an antibodyagainst the jS-subunit was used for immunoblotting, twobands, presumably the aj8y-trimeric complex and the /3y-dimer, were detected (Fig. 6B, lane 2) in race 4 IF-treatedpreparation. The upper band detected by anti-Gsa and

anti-Gp appeared to have the same location on the gel,whereas the lower bands cross-reacted with anti-Gsot andanti-Gp appeared at different positions. Only the upperband was detected in treatment with no IF or with race5 IF (Fig. 6B, lanes 1 and 3).

Discussion

G proteins have been widely studied as mediators ofcellular signalling processes and many G protein coupledreceptors have been identified (Kaziro et al., 1991). Signaltransducing G proteins are classified largely into twogroups, i.e. heterotrimeric G proteins and low-molecular-weight monomeric G proteins (Hall, 1990; Gilman, 1987).The two classes of G proteins differ in their response tonon-hydrolysable GTP analogues (Bourne, 1988) orfluoride ions (Higashijima et al., 1987; Kahn, 1991).Whereas the functions regulated by trimeric G proteinsare stimulated by non-hydrolysable analogues of GTP,the effector functions mediated by the monomeric Gproteins are inhibited (Iyengar and Birnbaumer, 1990).Fluoride is a potent, rapidly reversible activator of thetrimeric G proteins. As a result, numerous G protein-mediated signal transduction pathways are stimulated inmembrane preparations or crude cell lysates by fluorideions. Activation of purified G proteins by non-hydrolysingGTP analogues (e.g. GTPyS) or by A1F4~ results ina change in the conformation of the Ga subunit(Higashijima et al., 1987) which promotes its dissociationfrom the trimeric complex and its association with the

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effector(s) (Northup et al., 1983a, b). Compelling evidenceindicates that fluoride acts as an aluminium complex,A1F4~ (Higashijima et al., 1987; Sternweis and Gilman,1982) that mimics the y-phosphate of GTP when boundto the GDP-bound form of Ga (Bigay et al., 1985, 1987).

The treatment of plasma membranes with IFs of race2.3 and race 4 (incompatible on plants with the Cf-5gene) resulted in the dephosphorylation of the H + -ATPase, whereas no dephosphorylation was observedwhen the membranes were treated with IFs of race 5 andrace 2.4.5.9.11 (compatible on plants with the Cf-5 gene)(Fig. 1). When plasma membranes were treated with noIF or with a 1:160 dilution of race 4 IF, the plasmamembrane H+-ATPase was not dephosphorylated, butdephosphorylation occurred if GTP(y)S or A1F4~ wereincluded in the reaction (Fig. 2), supporting the role oftrimeric G proteins in mediating the elicitor-induced H + -ATPase dephosphorylation.

To identify the subtype of the trimeric G proteinfurther, three different bacterial toxins were tested in thephosphorylation reaction. In contrast to A1F4" which isa universal reversible activator of the trimeric G proteinfamily, these bacterial toxins utilize distinctive subclassesof G proteins as substrates for covalent modification,exerting their effects by selectively ADP-ribosylatingspecific subtypes of G proteins. The cholera toxin, CTX,ADP-ribosylates Gs, retaining it in the GTP bound form(Gill and Meren, 1978; Cassel and Selinger, 1977). Thepurtessis toxin, PTX, ADP-ribosylates the Gj family andGo in the GDP bound form, preventing GTP binding(Dolphin, 1987). The botulinum toxin, toxin C3, ADP-ribosylates monomeric G proteins, but not the ras geneproduct (Rubin et al., 1988). These toxins can be usedto interfere with the signal transduction process in eitherintact cells or cell-free systems. CTX induced the dephos-phorylation of the plasma membrane H + -ATPase in theabsence of active elicitors whereas PTX or toxin C3 hadno effect on dephosphorylation (Fig. 3). Moreover, theincubation of plasma membranes with CTX resulted inthe ADP-ribosylation of a 42 kDa polypeptide, a reactionthat was inhibited by pre-incubation of the membraneswith antibodies raised against the a-subunit of stimulatoryG proteins (Fig. 4). Further experiments with antibodiesraised against the a-subunit of different trimeric G proteinsubtypes revealed that the elicitor-induced dephosphoryl-ation of H+-ATPase was blocked by pre-incubatingplasma membranes with the antibody against Gsa,whereas antibodies against G1CI-1, Giot-2, Giot-3 or Go didnot affect the dephosphorylation (Fig. 5A). The Gsot

antibody also cross-reacted with a 42 kDa polypeptide(Fig. 5B).

It should be noted that the size of plant proteinsrecognized by antibodies raised against mammalian a-subunits of G proteins ranges from 30 kDa (Warpehaet al., 1991) to 50 kDa (Jacobs et al., 1988). In animals

Gs activation by fungal elicitors 235

and yeast, the molecular mass of this subunit is alwaysabout 40 kDa. Since the proteins recognized by theseantibodies in plants have not been isolated and sequenced,the specificity of the recognition remains to be proven.To date, several cDNAs and/or genes encoding G proteinsin plants have been cloned, generally by screening cDNAor genomic libraries with oligonucleotide probes deducedfrom mammalian G proteins. Two related Go subunits,one from Arabidopsis and one from tomato, have beencloned (Ma et al., 1990, 1991). The predicted molecularmass of the a-subunit of this tomato G protein is 44.9 kDa(Ma et al., 1991), which is in agreement with the estimatedmolecular mass of 42 kDa (based on SDS-PAGE)reported in this work. This tomato G protein sequence,although still not designated to type, includes all of theconserved regions characteristic of known Ga, as well asconsensus myristoylation and CTX ADP-ribosylationsites (Ma et al., 1991).

It is assumed that such G proteins are attached to theinner face of the plasma membrane (Gilman, 1987). Thenature of this attachment facilitates the isolation of nativeand active G proteins without disturbing the existingsubunit status. Thus, a protocol designed to 'wash' thesubunits from inside-out plasma membrane vesicles afterelicitor treatment was used. After elicitating plasma mem-branes with IF preparations from race 4 C. fulvum, twoproteins were detected on immunoblots after native PAGEby an antibody raised against Gsa (Fig. 6A). The proteinwith lower molecular weight may represent the a-subunitafter elicitor-induced subunit dissociation. The proteinwith higher molecular weight may represent the non-dissociated G protein or the re-associated ajSy-complex.When an antibody raised against the j3-subunit was usedin immunoblots, two bands were detected in race 4IF-treated preparations (Fig. 6B). With untreated, or race5 IF-treated membrane vesicles, only the high-molecular-weight protein could be detected by either of the antibod-ies (Fig. 6). These observations suggest that subunits ofa stimulatory trimeric G protein undergo a dissociationprocess upon elicitation.

It has been proposed (Keen, 1990; De Wit, 1992) thata pathogen elicitor interacts with a receptor, the putativeproduct of a resistance gene, located on the outsidesurface of the host plasma membrane. The tomato Cf-9gene, which confers resistance to infection by races of C.fulvum that carry the avr9, was isolated (Jones et al.,1994). The DNA sequence of Cf-9 encodes a putativetransmembrane protein with an extracellular domaincomposed of 28 leucine-rich repeats (LRR) and a shortcytoplasmic tail. The LRR motif is a receptor modulethat has been related to several different cytoplasmicsignalling mechanisms (Jones et al., 1994). Although Cf-9 lacks any obvious cytoplasmic signalling capacity, itcould generate a signal by the interaction between itstransmembrane domain or cytoplasmic tail and a cyto-

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236 Xing et al.

plasmic signalling mechanism. A protein with homologyto the receptor-like kinase (RLK) catalytic domain con-ferring resistance to races of Pseudomonas syringae pv.tomato that carry the avirulence gene avrPto has beenidentified. Pto encodes a serine-threonine protein kinasethat might be associated with the plasma membrane(Martin et al., 1993). The activation of a protein kinaseC-like kinase has previously been reported in Cf-5 cellstreated with elicitors isolated from race 4 of C. fulvum(Xing et al., 1996). However, the putative receptorencoded by Cf-9 gene lacks the protein kinase domain ofRLKs (Jones et al., 1994). These results would suggestthe absence of a direct coupling between the binding ofthe elicitor to the receptor and the protein kinase activityin our experimental system. The present work suggeststhat upon binding of the elicitor to the putative receptor,a trimeric G protein is activated, promoting the bindingof GTP to the a-subunit that dissociates from the |8y-complex and interacts with an effector. This is followedby hydrolysis of GTP and the subsequent return to thetrimeric conformation.

In conclusion, these results demonstrate the activationof a stimulatory G protein (Gs) by fungal elicitors. TheG protein, in turn, transduces the signal by activating amembrane-bound phosphatase that mediates the dephos-phorylation of the host plasma membrane H+-ATPase.Previous work in this laboratory has shown that after theelicitor-induced dephosphorylation of the H+-ATPase inintact cells, the enzyme is rephosphorylated by two proteinkinases (a protein kinase C-like and a Ca2+/CaM-depend-ent kinase) (Xing et al., 1996). Whether these processesare mediated by other trimeric or monomeric G proteinsis currently under investigation.

Acknowledgements

The authors thank Professor Ramon Serrano (Valencia, Spain)for supplying the antibodies raised against the H + -ATPasefrom Arabidopsis. This research was supported by grants fromthe Natural Sciences and Engineering Research Council ofCanada to VJH and EB.

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